PROJECT SUMMARY It has been proposed that predation by virulent (lytic) phages on Vibrio cholerae in aquatic habitats plays a causal role in terminating cholera epidemics. However, there has been no systematic analysis of phage predation in such habitats to know whether this model holds water or not. We found that, of three virulent phages that are highly prevalent in Bangladesh during epidemics, two, ICP2 and ICP3, are incapable of killing or multiplying on V. cholerae in fresh or estuarine water environments. In contrast, both phages are able to replicate on V. cholerae during infection of the small intestine, suggesting that is their primary niche. The third phage, ICP1, which is a larger, more complex phage, is able to multiply and kill V. cholerae in the small intestine and in estuarine environments, though not in fresh water. Because fresh water is the primary vehicle for water-borne transmission of cholera during outbreaks, our data argue against a role for phage predation in the environment in terminating outbreaks. On the other hand, the intriguing ability of ICP1 to prey on estuarine V. cholerae, including slowly-growing and nongrowing dormant cells suggests this phage may play an important role in the population dynamics of V. cholerae in its permanent reservoir, namely the estuaries surrounding the Bay of Bengal. The ability of ICP1 to prey on estuarine V. cholerae may also be a major factor contributing to the high prevalence of ICP1 in both the environment and in cholera patient rice-water stools in Bangladesh. To gain a better understanding of ICP1?s ability to prey on estuarine V. cholerae, in this exploratory project we propose to test the hypothesis that ICP1 harbors a set of genes that are needed specifically for predation on slowly or nongrowing V. cholerae but not on V. cholerae growing in rich media or in the small intestine. We propose to use transposon-sequencing (Tn-seq) to identify and characterize these genes in order to reveal the mechanisms of phage predation in estuarine environments.